Modeling coronavirus spike protein dynamics: implications for immunogenicity and immune escape

The ongoing COVID-19 pandemic is a global public health emergency requiring urgent development of efficacious vaccines. While concentrated research efforts have focused primarily on antibody-based vaccines that neutralize SARS-CoV-2, and several first-generation vaccines have either been approved or received emergency use authorization, it is forecasted that COVID-19 will become an endemic disease requiring updated second-generation vaccines. The SARS-CoV-2 surface spike (S) glycoprotein represents a prime target for vaccine development because antibodies that block viral attachment and entry, i.e., neutralizing antibodies, bind almost exclusively to the receptor-binding domain. Here, we develop computational models for a large subset of S proteins associated with SARS-CoV-2, implemented through coarse-grained elastic network models and normal mode analysis. We then analyze local protein domain dynamics of the S protein systems and their thermal stability to characterize structural and dynamical variability among them. These results are compared against existing experimental data and used to elucidate the impact and mechanisms of SARS-CoV-2 S protein mutations and their associated antibody binding behavior. We construct a SARS-CoV-2 antigenic map and offer predictions about the neutralization capabilities of antibody and S mutant combinations based on protein dynamic signatures. We then compare SARS-CoV-2 S protein dynamics to SARS-CoV and MERS-CoV S proteins to investigate differing antibody binding and cellular fusion mechanisms that may explain the high transmissibility of SARS-CoV-2. The outbreaks associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 over the last two decades suggest that the threat presented by coronaviruses is ever-changing and long term. Our results provide insights into the dynamics-driven mechanisms of immunogenicity associated with coronavirus S proteins and present a new, to our knowledge, approach to characterize and screen potential mutant candidates for immunogen design, as well as to characterize emerging natural variants that may escape vaccine-induced antibody responses.     

基于新型冠状病毒S蛋白结构及入胞机制的抗体与药物研发

新型冠状病毒肺炎,世界卫生组织命名为“2019冠状病毒病”（corona virus disease 2019, COVID-19）,是一种由2019新型冠状病毒（2019 nCov）感染导致的肺炎。目前新冠肺炎在全球广泛流行,且疫情尚未得到全部控制。由于新型冠状病毒表面的刺突蛋白（spike protein,S）介导病毒与细胞膜受体结合并参与入胞过程,S蛋白在病毒的传播过程中发挥着重要作用。针对S蛋白的研究不仅可以解析病毒相关蛋白质结构与功能,阐释其入胞机制,同时也为新冠肺炎的预防、诊断与治疗提供相关信息,有着重要的应用价值。S蛋白与特异性受体--血管紧张素酶II（angiotensin converting enzyme II, ACE2）结合,相较于SARS病毒,新型冠状病毒S蛋白的RBD区域（receptor binding domain）与ACE2亲和力更高,但其S蛋白与ACE2结合能力整体上弱于SARS病毒。S蛋白结合ACE2受体 介导的新型冠状病毒入胞机制包括胞吞和非胞吞途径。丝氨酸蛋白酶2（transmembrane protease serine 2, TMPRSS2）、溶酶体组织蛋白酶（lysosomal cathepsin）和Furin蛋白酶可切割S蛋白S1和S2亚基间的酶切位点,促进病毒和靶膜的融合。基于S蛋白的结构,本文从抗体的结合位点、来源与类型等方面对靶向新型冠状病毒S蛋白的抗体进行了比较分析,对相关药物作用机制与进展进行了综述。虽然靶向冠状病毒S蛋白的抗体和药物特异性高,治疗效果较好,但部分试剂的作用机制、安全性、适用性和稳定性等性质仍未研究透彻,需要严格评估,因此其研发与应用也存在着一定挑战。     

Structures and polymorphic interactions of two heptad-repeat regions of the SARS virus S2 protein

Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct alpha-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.     

Epigenetic regulation of autophagy in coronavirus disease 2019 (COVID-19)

The coronavirus disease 2019 (COVID-19) pandemic has become the most serious global public health issue in the past two years, requiring effective therapeutic strategies. This viral infection is a contagious disease caused by new coronaviruses (nCoVs), also called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autophagy, as a highly conserved catabolic recycling process, plays a significant role in the growth and replication of coronaviruses (CoVs). Therefore, there is great interest in understanding the mechanisms that underlie autophagy modulation. The modulation of autophagy is a very complex and multifactorial process, which includes different epigenetic alterations, such as histone modifications and DNA methylation. These mechanisms are also known to be involved in SARS-CoV-2 replication. Thus, molecular understanding of the epigenetic pathways linked with autophagy and COVID-19, could provide novel therapeutic targets for COVID-19 eradication. In this context, the current review highlights the role of epigenetic regulation of autophagy in controlling COVID-19, focusing on the potential therapeutic implications.     

DNA vaccination induced protective immunity against SARS CoV-2 infection in hamsterss

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19.     

Structure-guided antibody cocktail for prevention and treatment of COVID-19

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.     

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.     

Antibodies at work in the time of severe acute respiratory syndrome coronavirus 2

In view of devastating effects of COVID-19 on human life, there is an urgent need for the licened vaccines or therapeutics for the SARS-CoV-2 infection. Age-old passive immunization with protective antibodies to neutralize the virus is one of the strategies for emergency prophylaxis and therapy for coronavirus disease 2019 (COVID-19). In this review, the authors discuss up-to-date advances in immune-based therapy for COVID-19. The use of convalescent plasma therapy as the first line of defense to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been established, with encouraging results. Monoclonal antibodies (mAbs) that bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or block the interaction between SARS-CoV-2 RBD and the human angiotensin-converting enzyme 2 receptor have been found to be very promising as a countermeasure for tackling the SARS-CoV-2 infection, and clinical trials are underway. Considering the counterproductive antibody-dependent enhancement of the virus, mAbs therapy that is safe and efficacious, even in people with underlying conditions, will be a significant breakthrough. In addition, emerging immunotherapeutic interventions using nanobodies and cellular immunotherapy are promising avenues for tackling the COVID-19 pandemic. The authors also discuss the implication of mAbs as mediators of cytokine storm syndrome to modify the immune response of COVID-19 patients, thus reducing the fatality rate of COVID-19 infection.     

Facing the wrath of enigmatic mutations: a review on the emergence of severe acute respiratory syndrome coronavirus 2 variants amid coronavirus disease-19 pandemic

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging respiratory virus responsible for the ongoing coronavirus disease 19 (COVID-19) pandemic. More than a year into this pandemic, the COVID-19 fatigue is still escalating and takes hold of the entire world population. Driven by the ongoing geographical expansion and upcoming mutations, the COVID-19 pandemic has taken a new shape in the form of emerging SARS-CoV-2 variants. These mutations in the viral spike (S) protein enhance the virulence of SARS-CoV-2 variants by improving viral infectivity, transmissibility and immune evasion abilities. Such variants have resulted in cluster outbreaks and fresh infection waves in various parts of the world with increased disease severity and poor clinical outcomes. Hence, the variants of SARS-CoV-2 pose a threat to human health and public safety. This review enlists the most recent updates regarding the presently characterized variants of SARS-CoV-2 recognized by the global regulatory health authorities (WHO, CDC). Based on the slender literature on SARS-CoV-2 variants, we collate information on the biological implications of these mutations on virus pathology. We also shed light on the efficacy of therapeutics and COVID-19 vaccines against the emerging SARS-CoV-2 variants.     

Caffeic acid derivatives (CAFDs) as inhibitors of SARS-CoV-2: CAFDs-based functional foods as a potential alternative approach to combat COVID-19

BackgroundSARS-CoV-2, an emerging strain of coronavirus, has affected millions of people from all the continents of world and received worldwide attention. This emerging health crisis calls for the urgent development of specific therapeutics against COVID-19 to potentially reduce the burden of this emerging pandemic.PurposeThis study aims to evaluate the anti-viral efficacy of natural bioactive entities against COVID-19 via molecular docking and molecular dynamics simulation.MethodsA library of 27 caffeic-acid derivatives was screened against 5 proteins of SARS-CoV-2 by using Molegro Virtual Docker 7 to obtain the binding energies and interactions between compounds and SARS-CoV-2 proteins. ADME properties and toxicity profiles were investigated via www.swissadme.ch web tools and Toxtree respectively. Molecular dynamics simulation was performed to determine the stability of the lead-protein interactions.ResultsOur obtained results has uncovered khainaoside C, 6-O-Caffeoylarbutin, khainaoside B, khainaoside C and vitexfolin A as potent modulators of COVID-19 possessing more binding energies than nelfinavir against COVID-19 M^pro, Nsp15, SARS-CoV-2 spike S2 subunit, spike open state and closed state structure respectively. While Calceolarioside B was identified as pan inhibitor, showing strong molecular interactions with all proteins except SARS-CoV-2 spike glycoprotein closed state. The results are supported by 20 ns molecular dynamics simulations of the best complexes.ConclusionThis study will hopefully pave a way for development of phytonutrients-based antiviral therapeutic for treatment or prevention of COVID-19 and further studies are recommended to evaluate the antiviral effects of these phytochemicals against SARS-CoV-2 in in vitro and in vivo models.     

ACE2, B0AT1, and SARS-CoV-2 spike protein: Structural and functional implications

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a public health crisis and led to tremendous economic devastation. The spike protein (S) of SARS-CoV-2 hijacks the angiotensin converting enzyme 2 (ACE2) as a receptor for virus entry, representing the initial step of viral infection. S is one of the major targets for development of the antiviral drugs, antibodies, and vaccines. ACE2 is a peptidase that plays a physiologically important role in the renin–angiotensin system. Concurrently, it also forms dimer of heterodimer with the neutral amino acid transporter B⁰AT1 to regulate intestinal amino acid metabolism. The symptoms of COVID-19 are closely correlated with the physiological functions of ACE2. In this review, we summarize the functional and structural studies on ACE2, B⁰AT1, and their complex with S of SARS-CoV-2, providing insights into the various symptoms caused by viral infection and the development of therapeutic strategies.     

Screening of inhibitors against SARS-CoV-2 spike protein and their capability to block the viral entry mechanism: A viroinformatics study

SARS-CoV-2, previously named 2019 novel coronavirus (2019-nCoV), has been associated with the global pandemic of acute respiratory distress syndrome. First reported in December 2019 in the Wuhan province of China, this new RNA virus has several folds higher transmission among humans than its other family member (SARS-CoV and MERS-CoV). The SARS-CoV-2 spike receptor-binding domain (RBD) is the region mediating the binding of the virus to host cells via Angiotensin-converting enzyme 2 (ACE2), a critical step of viral. Here in this study, we have utilized in silico approach for the virtual screening of antiviral library extracted from the Asinex database against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein. Further, the molecules were ranked based on their binding affinity against RBD, and the top 15 molecules were selected. The affinity of these selected molecules to interrupt the ACE2-Spike interaction was also studied. It was found that the chosen molecules were demonstrating excellent binding affinity against spike protein, and these molecules were also very effectively interrupting the ACE2-RBD interaction.Furthermore, molecular dynamics (MD) simulation studies were utilized to investigate the top 3 selected molecules' stability in the ACE2-RBD complexes. To the best of our knowledge, this is the first study where molecules' inhibitory potential against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein and their inhibitory potential against the ACE2-Spike has been studied. We believe that these compounds can be further tested as a potential therapeutic option against COVID-19.     

SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using platelet-poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to β and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.     

The flexibility of ACE2 in the context of SARS-CoV-2 infection

The coronavirus disease 2019 (COVID-19) pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine development efforts are underway, there are many outstanding questions on the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and host cell entry. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular spike glycoprotein and at the receptor-binding domain (RBD)-receptor interface, suggesting a role in infection. Here, we perform explicitly solvated, all-atom, molecular dynamics simulations of the glycosylated, full-length, membrane-bound ACE2 receptor in both an apo and spike RBD-bound state to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full-length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer and suggests a mechanical contribution of the host receptor toward the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can potentially be exploited for the rational design of effective SARS-CoV-2 therapeutics.     

Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.     

Chloroquine paradox may cause more damage than help fight COVID-19

Novel coronavirus disease 2019 (COVID-19) pandemic is the most recent health care crisis without specific prophylactic or therapeutic drugs. Antimalarial drug chloroquine (CHL) and its safer derivative hydroxychloroquine (HCHL) have been proposed to be repurposed to treat SARS coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19. CHL/HCHL have anti-inflammatory activity and are used to treat rheumatoid arthritis, osteoarthritis and lupus. Although, CHL/HCHL have an anti-viral activity against several viruses in cell-cultures, the anti-viral activity in-vivo is questionable. Repurposing of CHL/HCHL to treat SARS-CoV-2 infection is appealing. However, there is empirical evidence from animal studies with other viruses suggesting that CHL/HCHL may have an untoward paradoxical effect. One thus cannot exclude the possibility that CHL may increase the severity of the disease and prove deleterious both for the patients and public health efforts to contain the highly contagious and explosive spread of SARS-CoV-2.     

A systematic review of Vaccine Breakthrough Infections by SARS-CoV-2 Delta Variant

Vaccines are proving to be highly effective in controlling hospitalization and deaths associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as shown by clinical trials and real-world evidence. However, a deadly second wave of coronavirus disease 2019 (COVID-19), infected by SARS-CoV-2 variants, especially the Delta (B.1.617.2) variant, with an increased number of post-vaccination breakthrough infections were reported in the world recently. Actually, Delta variant not only resulted in a severe surge of vaccine breakthrough infections which was accompanied with high viral load and transmissibility, but also challenged the development of effective vaccines. Therefore, the biological characteristics and epidemiological profile of Delta variant, the current status of Delta variant vaccine breakthrough infections and the mechanism of vaccine breakthrough infections were discussed in this article. In addition, the significant role of the Delta variant spike (S) protein in the mechanism of immune escape of SARS-CoV-2 was highlighted in this article. In particular, we further discussed key points on the future SARS-CoV-2 vaccine research and development, hoping to make a contribution to the early, accurate and rapid control of the COVID-19 epidemic.     

新型冠状病毒抗体和小分子抑制剂的研究现状

世界卫生组织已宣布新型冠状病毒感染（coronavirus disease 2019,COVID-19）的爆发为全球大流行。中和抗体和小分子抑制剂在预防及治疗COVID-19中发挥重要作用。尽管已开发出了多种中和抗体以及疫苗,但是随着病原体严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的不断变异,现有的抗体及疫苗面临巨大的挑战。小分子抑制剂主要通过干扰病毒与宿主的结合以及病毒自身的复制达到消灭病毒以及抑制病毒感染的作用,并且对SARS-CoV-2突变株具有广谱抑制作用,是当前研究的热点。近年来国内外学者对SARS-CoV-2的小分子抑制剂做了大量的研究工作,本文根据中和抗体识别的抗原表位以及小分子抑制剂的作用机制分别对用于预防及治疗COVID-19的中和抗体和小分子抑制剂进行综述,讨论其研究现状,并展望小分子抑制剂的应用前景,以期为该领域的进一步研究提供参考。     

SARS-CoV-2 VOCs,Mutational diversity and clinical outcome: Are they modulating drug efficacy by altered binding strength?

The global COVID-19 pandemic continues due to emerging Severe Acute Respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC). Here, we performed comprehensive analysis of in-house sequenced SARS-CoV-2 genome mutations dynamics in the patients infected with the VOCs - Delta and Omicron, within Recovered and Mortality patients. Statistical analysis highlighted significant mutations - T4685A, N4992N, and G5063S in RdRp; T19R in NTD spike; K444N and N532H in RBD spike, associated with Delta mortality. Mutations, T19I in NTD spike, Q493R and N440K in the RBD spike were significantly associated with Omicron mortality. We performed molecular docking for possible effect of significant mutations on the binding of Remdesivir. We found that Remdesivir showed less binding efficacy with the mutant Spike protein of both Delta and Omicron mortality compared to recovered patients. This indicates that mortality associated mutations could have a modulatory effect on drug binding which could be associated with disease outcome.     

High-throughput,single-copy sequencing reveals SARS-CoV-2 spike variants coincident with mounting humoral immunity during acute COVID-19

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH₂-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.